medium no. 66: modified Murashige and Skoog medium, 0.2% (w/v) gellan gum, pH 5.7

Plant cell lines

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • Fumalic acid

  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)

  • D(+)-Glucose

  • MSK_VT

    Chemical

    Concentration (mg/mL)

    Pyridoxine·HCl

    1

    Thiamine·HCl

    1

    myo-Inositol

    100

    L-Ascorbic acid

    2

    Calcium (+)-pantothenate

    1

    Choline chloride

    1

    Nicotinamide

    1

    Folic acid

    0.4

    Riboflavin

    0.2

    p-Aminobenzoic acid

    0.02

    Cyanocobalamin

    0.02

    (+)-Biotin

    0.02

    Store at −20°C

  • MSK_F

    Chemical

    Concentration (mg/mL)

    Citric acid monohydrate

    43.8

    DL-Malic acid

    40

    Sodium pyruvate

    20

    Store at −20°C

  • MSK_G

    Chemical

    Concentration (mg/mL)

    D(−)-Fructose

    2.5

    D(+)-Cellobiose

    2.5

    D(−)-Mannitol

    2.5

    D(+)-Mannose

    2.5

    α-L(+)-Rhamnose monohydrate

    2.77

    D(−)-Ribose

    2.5

    D(−)-Sorbitol

    2.5

    D(+)-Xylose

    2.5

    Store at −20°C

  • Gellan gum

    Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)

  • NaOH (5 N)

Medium preparation

  1. Dissolve the following chemicals in approximately 300 mL of distilled water with NaOH (5 N).

    Chemical

    Amount

    Fumalic acid

    2.04 g

    NaOH (5 N)

    6 mL

  2. Fill up to approximately 800 mL with distilled water, and dissolve the following chemicals.

    Chemical

    Amount

    MS salt mix

    1 bag (1 L)

    D(+)-Glucose

    40 g

  3. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution

    Volume (mL)

    MSK_VT

    1

    MSK_F

    1

    MSK_G

    1

  4. Adjust the pH of the solution to 5.7 with NaOH (5 N), and fill up to 1 L with distilled water.

  5. Add 2 g of gellan gum to the medium.

  6. Autoclave the medium at 121°C for 20 min.

Formulation

Chemical

Concentration (mg/L)

KNO3

1900

NH4NO3

1650

CaCl2·2H2O

440

MgSO4·7H2O

370

KH2PO4

170

H3BO3

6.2

MnSO4·4H2O

22.3

ZnSO4·7H2O

8.6

KI

0.83

Na2MoO4·2H2O

0.25

CuSO4·5H2O

0.025

CoCl2·6H2O

0.025

FeSO4·7H2O

27.8

Na2-EDTA

37.3

Pyridoxine·HCl

1

Thiamine·HCl

1

myo-Inositol

100

L-Ascorbic acid

2

Calcium (+)-pantothenate

1

Choline chloride

1

Nicotinamide

1

Folic acid

0.4

Riboflavin

0.2

p-Aminobenzoic acid

0.02

Cyanocobalamin

0.02

(+)-Biotin

0.02

Fumalic acid

2040

Citric acid monohydrate

43.8

DL-Malic acid

40

Sodium pyruvate

20

D(+)-Glucose

40000

D(−)-Fructose

2.5

D(+)-Cellobiose

2.5

D(−)-Mannitol

2.5

D(+)-Mannose

2.5

α-L(+)-Rhamnose monohydrate

2.77

D(−)-Ribose

2.5

D(−)-Sorbitol

2.5

D(+)-Xylose

2.5

Gellan gum

2000